High CD38 expression defines a mitochondrial function adapted CD8 + T cell subset with implications for lung cancer immunotherapy

doi:10.21203/rs.3.rs-4815459/v1

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High CD38 expression defines a mitochondrial function adapted CD8 + T cell subset with implications for lung cancer immunotherapy

https://doi.org/10.21203/rs.3.rs-4815459/v1

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Despite identifying specific CD8⁺T cell subsets associated with immunotherapy resistance, the molecular pathway triggering the process remains elusive. Given the potential of CD38 in regulating CD8⁺T cell function, we aimed to observe the accumulation of CD38⁺CD8⁺T cells in lung cancer and further explored its role in immunotherapy resistance. Phenotypic analysis of tumoral CD8⁺T cells from both lung cancer patients and immunotherapy-resistant pre-clinical models identifies that CD38-expressing CD8⁺T cells displayed as CD38^hi and CD38^intT cell subsets. Following, it was observed a higher expression of CD38 along with T cell exhaustion genes and dysregulated mitochondrial bioenergetics. In addition, it was suggested that an evaluated CD38^hiCD8⁺T cells in peripheral but not in center of TME were associated with good response to anti-PD-1 therapy in NSCLC, as well as corresponding depth of clinical regression, which was evidenced by more depletion of CD38^hiCD8⁺T cells occur in subject with higher regional CD38^hiCD8⁺T cells infiltration. As expected, it was found that ICIs-resistant murine lung cancer models had lack of effective reduction in term of CD38^hiCD8⁺T cells when receiving PD-L1 mAb alone. Notably, combination therapy of PD-L1 mAb and EGCG could selectively restrict CD38^hiCD8⁺T cells infiltration and enhance IFN-γ production by these T cells, thereby significantly improved survival in this carcinoma model. This restored immunotherapy sensitivity was found to be related to the selective improved mitochondrial of CD38^hiCD8⁺T cells, which was validated by the established link between IFN-γ production and mitochondrial metabolism. Collectively, our data highlighted a role for the CD38-coupled dysfunctional mitochondrial in promoting CD8⁺T cell exhaustion and intrinsic resistance to ICIs therapy, thereby offered a rationale target to enhance the therapeutic efficacy of PD-1 blockade therapy in lung cancer.

CD38

PD-1

CD8 + T cell

Tumor microenvironment

lung cancer

Immunotherapy

mitochondrion

Non-small-cell lung cancer (NSCLC) is a common malignant tumor causing a leading morbidity and mortality worldwide. Especially in China, the incidence and fatality rate of NSCLC are on the top of the list and still show an upward trend in recent years^[1]. Although there are multidisciplinary treatments, such as surgery, chemotherapy, radiotherapy, and targeted drug therapy for NSCLC, its overall survival rate is still unsatisfactory^[2–4]. Therefore, it is necessary to identify potential target molecules or target cells and developing new therapeutic strategies that can improve the clinical benefits of NSCLC patients.

Although anti-PD-1 has revolutionized the treatment for several NSCLC subtypes in recent years, only around 20% of patients respond to immune checkpoint blockade (ICB)^[5]. A proposed mechanism for a lack of response to ICB is that NSCLC with insufficient tumor infiltration of tumor-specific T cells may be refractory to treatment^[6]. The combination of ICB with another treatment modality capable of stimulating tumor-specific T cell responses in the tumor microenvironment is a potential strategy to increase responses to ICB^[7]. With the crucial capability of directly killing tumor cells, tumor-infiltrating CD8⁺T cells have been suggested to be effectively activated by anti-PD-1 blockade, and consequently dominate immunotherapy mechanism in NSCLC^[8]. However, it is still unclear which subgroups of CD8⁺T cells play such vital roles in immunotherapy. Previous studies have identified several subgroups of CD8⁺T cells responsible of anti-tumor effect in tumor-killing process^[9], which implicated our current exploration on whether the subgroup of CD38⁺CD8⁺T cells also act as a member involved in tumor-killing process, in the hope of helping improve the success rate of immunotherapy for lung cancer patients^[10].

CD38 is a member of the ribosyl cyclase family that is widely expressed on the surface of nonhematopoietic cells and diverse immune cells^{[11, 12]}. Both its receptor-ligand and enzymatic functions are implicated in tumorigenesis and tumor progression by modulating immune regulation, metabolism, calcium-mediated signal transduction, cell adhesion, and migration^[13]. The growing body of evidence implicating CD38 in tumor immune evasion led us to hypothesis that CD38 expression on lung cancer tumor-infiltrating immune cells (TIICs) contributes to tumor progression and may serve as an important immunotherapeutic target^{[2, 14]}.

In present study, we describe a subset of lung cancer infiltrated CD8⁺T cells that are characterized by heterogeneous expression of ribose hydrolase CD38 and that regional CD38^hiCD8⁺T cells work as a target for anti-PD-1 therapy. Notably, modulated CD38^hiCD8⁺T cells, specifically mitochondrial function, drive the establishment of anti-tumor immunity induced by anti-PD-1 mAb. Together, these results imply that accumulation of CD38^hiCD8⁺T cells in context of distinct TME is a mechanism of intrinsic resistance to ICB, and should stimulate development of new therapeutic strategies.

2.1 Patients

Forty-two patients with lung cancer were rerolled from the First Affiliated Hospital of Soochow University from 2021 to 2023, and none of them had received anti-tumor therapy at the time of sample analysis. Clinic information included age, sex, and histological subtype. This study was approved by ethics committee of the First Affiliated Hospital of Soochow University.

2.2 Sample collection

A bronchoscope was used to attach the lung cancer lesion. Regarding visualized neoplasm under the bronchoscope, a superficial biopsy was performed (n = 29). For peribranchial lesions, intratumoral endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) with a 22-gauge needle was performed (n = 13). Written informed consent was obtained from all patients.

2.3 Mice and cell lines

6–8-week-old female C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were housed at 23 ± 2°C with specific pathogen-free conditions. Mice were fed food and water ad libitum. Mice lung carcinoma cell line (3LL) was kindly provided by Prof. Bin-feng Lu (University of Pittsburgh, PA, USA). The cell was cultured in RPMI1640 supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 mg/mL streptomycin, 100 U/mL penicillin, and 50 mM 2-ME. The cell was incubated at 37°C with 5% CO₂.

2.4 Mouse tumor experiments

3LL were injected intradermally into B6 mice, and the tumor volume was measured with a caliper every 2–3 days and calculated as follows: 0.5 × L × W² (L is the longest dimension and W is the perpendicular dimension). Tumor bearing mice were randomized into four treatment cohorts: (i) control IgG; (ii) anti-PD-L1 monoclonal antibodies (mAbs), (iii) EGCG (E4143, Sigma-Aldrich), (iv) anti-PD-L1 mAbs (10F.9G2, InVivoPlus) plus EGCG. All antibodies were administered at a dose of 150 µg /mouse through intraperitoneal injection twice per week. After the day the mice were injected with 3LL, they were orally administered EGCG at a dose of 50 mg/kg b.w. dissolved in 0.1 ml purified water every day. Mice were euthanized when the tumor volume reached 2000 mm³. The day of euthanasia was used to calculate survival.

2.5 Analysis of tumor-infiltrating lymphocytes (TILs)

Tumor tissues of humans and mice were dissected and placed in RPMI medium, then disrupted mechanically using scissors, digested with a mixture of DNase I (0.3 mg/ml, Sigma-Aldrich) and Liberase TL (0.2 mg/ml, Roche) in serum-free RPMI 1640 medium for 30 min, and dispersed through a 70-µm cell strainer (Beyotime Biotechnology). Flow cytometric analysis was performed using a FACS flow cytometer (FC500, Cytoflex, Beckman). All antibodies were purchased from Biolegend (USA). Combinations of the following fluorochrome-conjugated antibody (CD45, CD8, CD38, PD-1) were used for cell surface staining among defined population of CD45 + CD8 + T cells.

For ex vivo restimulation, freshly isolated single-cell suspension was cultured in complete RPMI 1640 medium containing PMA (50 ng/ml) and ionomycin (500 ng/ml) for 3 hours before it was analyzed for IFN-γ production by intracellular staining with IFN-γ mAbs (clone XMG1.2, Invitrogen). Multi-colored flow cytometry analyses were performed on Cytoflex (Beckman). Data were analyzed with Flowjo software (Tree Star).

2.6 Mitochondrial analysis

The single cells (10⁶ cells/well) were dyed with 100 nM of Mitotracker Green (MTG, Invitrogen), and 500 nM of Tetramethylrhodamine methyl ester (TMRM, Invitrogen), 500 nM of Rhod-2 (Invitrogen), 500 nM of MitoSOX Red (Invitrogen) in complete RPMI 1640 medium for 0.5h at 37◦C in 5% CO₂. After being washed, the cells were stained with the following antibodies: Zombie Red, APC-labeled anti-CD45 mAb (30-F11), APC-Cyanine7-labeled anti-CD8α mAb (53 − 6.7), PE-Cyanine7-labeled anti-PD-1 mAb (29F .1A12) for 30 min at 4°C in the dark. Thermo-life Attune NxT was immediately used for analysis. Multi-colored flow cytometry was performed on Cytoflex (Beckman) and data were analyzed with FlowJo software (Tree Star).

2.7 In vitro blockade culture

Human lung cancer tissues were placed in RPMI1640 containing 5% fetal bovine serum and 1% double antibiotic and transported back to the laboratory immediately at 4 ◦C immediately. After removing necrotic tissue, blood clots and other materials, the tumor tissues were washed several times with PBS, then cut into 1 mm² fragments in the culture medium. Appropriate amount of 0.1% collagenase IV was added, and the mixture was incubated in a shaker water bath at 37◦C. After 30 min, the culture medium was added to terminate digestion. The cell suspension was filtered with a 100µm cell strainer, centrifuged for 5 min, and the supernatant was discarded. The cells were resuspended with culture medium and randomized into two treatment cohorts: (i) control IgG; (ii) human anti-PD-1 mAb (10 µg/ml, Biolegend). After incubation at 37◦C with 5% CO₂ for 72 hours, fluorochrome-conjugated CD45, CD8 and CD38 mAb were used for staining among these two groups of cells by flow cytometric analysis.

2.8 Immunofluorescent Analysis

Formalin-fixed paraffin-embedded (FFPE) patient tissue samples from resected lung cancer were obtained from the First Affiliated Hospital of Soochow University (n = 40). FFPE tissue sections (5 µm) were deparafFinized in xylene and rehydrated in decreasing concentrations of ethanol (100, 90, 70, and 50% and distilled water; 5 min each). For antigen restoration, the sections were immersed in sodium citrate buffer (10mM, pH 5.5) and microwaved. After this, sections were washed 3x in Phosphate Buffered Saline with Tween-20 (PBST). To block nonspecific binding, sections were incubated with 3% bovine serum albumin (BSA) for 1 h at 3◦C. Following this, they were stained with rabbit anti-human CD8 mAb (1:600) and mouse anti-human CD38 (1:300) overnight at 4◦C. After washing with PBST, primary antibodies were detected with Abcam Cy3-conjugated goat anti- rabbit IgG (1:1000) and Abcam 488-conjugated goat anti- mouse IgG (1:1200). After washing, sections were subsequently embedded with DAPI. Sections were scanned using Nikon imaging system (Eclipse Ni-U). Mean fluorescence intensity (OD value) was quantified by ImageJ.

2.9 Assessment of Immunotherapy

Antitumor efficacy was assessed by reviewing computed tomography (CT) scans following two treatment courses. Patients who achieved a best response of Complete Response (CR)/Partial Response (PR) were termed responders and patients who achieved a best response of Stable Disease (SD)/Progressive Disease (PD) were termed non-responders. The same imaging modality was recommended for all patients. The change from baseline regarding the sum of target lesion diameters was measured according to RECIST 1.1.

2.10 Statistical analysis

Data (mean ± SEM) are representative of independent experiments. We used the two-tailed unpaired Student’s t-test, Mann-Whitney U test or the log-rank test (survival studies). The correlation analysis was evaluated with Pearson correlation and linear regression. Receiver operating characteristic (ROC) curves for response status were constructed to assess the prognostic ability of TILs. The association between response status and TIL frequency was analyzed using Fisher’s exact test. All analyses were two-sided and performed at a significance level of 5% (p < 0.05) using GraphPad 10.1.

3.1 CD38⁺CD8⁺T cell divided into CD38^hi and CD38^int subsets in human lung cancer

Firstly, to explore the role of CD38 in regulating T cell functionality, we carried out flow cytometry (FCM) analysis to identify CD38⁺CD8⁺T cells in lung cancer prior to any therapy. The baseline characteristics of the 42 patients with lung cancer are summarized in Table 1. Briefly, the median age was 67 years (range, 54–87 years), and histological analysis revealed 24 squamous cell carcinomas, 11 adenocarcinomas, and 7 small cell carcinomas.

As shown in Fig. 1a, the CD38 positive-CD8⁺T cells were commonly and prominently displayed as the two clusters, which were stratified as follows: cells with stronger CD38 expression (defined as CD38^hi cells), cells with median CD38 expression (defined as CD38^int cells). As shown in Fig. 1b, it was found that the percentages of infiltrated CD38^hiCD8⁺T cells, CD38^intCD8⁺T cells and CD38 negetive-CD8⁺T cells (CD38^negCD8⁺T cells) were comparable among lung cancer patients of different genders (37.05 ± 4.13% vs 32.92 ± 7.95%, 53.74 ± 7.04% vs 56.27 ± 2.53%, 7.39 ± 4.03% vs 9.46 ± 2.08%, p > 0.05, respectively), ages (29.02 ± 4.63% vs 40.11 ± 3.41%, 60.91 ± 3.87% vs 50.74 ± 3.06%, 7.91 ± 2.05% vs 7.53 ± 1.92%, p > 0.05, respectively). Meanwhile, the percentages of infiltrated CD38^hi/CD38^int/CD38^negCD8⁺T cells were also comparable among lung cancer of different pathologic types (NSCLC vs SCLC: 37.07 ± 3.15% vs 32.80 ± 6.81%, 50.71 ± 3.05% vs 59.10 ± 7.56%, 8.34 ± 1.56% vs 4.75 ± 1.76%, p > 0.05; Adeno vs Squamous: 29.24 ± 6.21% vs 40.48 ± 3.48%, 59.71 ± 4.73% vs 50.40 ± 3.09%, 8.12 ± 2.33% vs 8.37 ± 2.18%, p > 0.05).

3.2 Regional profile of CD38^hi/CD38^intCD8⁺ T cells in lung cancer microenvironments

To gain a more in-depth understanding of CD38 expression, we further investigated the infiltrated CD38^hi/CD38^intCD8⁺ T cell within distinct regions of tumor tissue. Superficial and intratumoral biopsy were used to sampled peripheral and central lung cancer tissue respectively (Fig. 2a). As shown in Fig. 2b, the ratio of CD38^hiCD8⁺T cells in the peripheral TME was greater than that in the central TME (43.89 ± 2.72% vs 20.88 ± 3.39%, p < 0.0001), while the ratio of CD38^intCD8⁺T cells in the peripheral TME was lower than that in the central TME (48.30 ± 2.66% vs 68.96 ± 4.52%, p < 0.0001).

To further confirm above findings, tumor sections of lung cancer were stained with anti-CD8 and anti-CD38 mAb, subsequently quantified by immunofluorescent microscope. As is shown in Fig. 2c, the data again indicated that the density of tumoral CD38⁺CD8⁺ T cells was higher in the peripheral TME than that in the central TME, which is similar with the result of flow cytometry. All findings demonstrated that CD38^hiCD8⁺T cells and CD38^intCD8⁺T cells displayed significant regional distribution, which suggested that the shift between CD38^hiCD8⁺T cells and CD38^intCD8 + T cells was involved in the TME heterogeneity.

3.3 Tumoral CD38^hiCD8 + T cells harbor more impaired mitochondria

NAD(H) and NADP(H) have traditionally been viewed as co-factors (or co-enzymes) involved in a myriad of oxidation-reduction reactions including the electron transport in the mitochondria^[15]. Considering CD38 is a key NAD(H)-dependent enzyme which breaks down NAD(H) to cyclic ADP-ribose (ADPR) and nicotinamide (NAM, vitamin B3), we examined mitochondrial mass, membrane potential, mitochondrial Ca²⁺ and reactive oxygen species level in CD38^hi/CD38^intCD8⁺T cells using the four indicators: MTG, TMRE, Rhod-2, and Mito SOX, respectively (Fig. 3). We observed that CD38^hiCD8⁺T cells have lower MTG expression compared to its CD38^int and CD38^neg counterparts (59.81 ± 3.13% vs 71.78 ± 3.38% vs 78.40 ± 3.71%). The TMRE expression was decreased in CD38^hiCD8⁺T cells (29.88 ± 3.43% vs 40.37 ± 4.30% vs 51.96 ± 5.01%). Increased Rhod-2 (70.68 ± 6.01% vs 59.24 ± 5.95% vs 45.34 ± 7.95%) and MitoSOX (60.66 ± 5.01% vs 44.43 ± 5.41% vs 29.36 ± 3.88%) was also observed in tumor-infiltrating CD38^hiCD8 + T cells. These data supported dysfunctional mitochondrial in CD38^hiCD8⁺ T cells compared with those responding CD38^intCD8⁺ T cell and CD38^negCD8⁺T cell, with evidence of defective function and compromised activity.

3.4 CD38^hiCD8⁺T cells displayed a more exhausted phenotype

PD-1 was well-known as an essential checkpoint molecule and widely used to define T cell exhaustion. To further explore the correlation of CD38 expression with those well-defined T cell exhaustion markers, we analyzed the expression of PD-1 in tumor-infiltrating CD38^hiCD8⁺ T cells and CD38^intCD8⁺T cells by flow cytometry (Fig. 4a). As shown in Fig. 4b, CD38^hiCD8⁺T cells expressed much higher level of PD-1, compared to their CD38^int counterparts (43.82 ± 2.68% vs 31.89 ± 2.21%, p < 0.01). Also, it was demonstrated this consistent pattern of PD-1 expression existed within both peritumoral (46.95 ± 43.52% vs 33.29 ± 32.59%, p < 0.05) and intratumoral compartments (46.70 ± 5.91% vs 28.73 ± 4.80%, p < 0.05).

Furthermore, we used Pearson’s correlations to examine the relationship between infiltrated CD38⁺CD8⁺T cells and PD-1⁺CD8⁺T cells in lung cancer TME. As shown in Fig. 4d, there is a strong positive relationship between infiltrated CD38^hiCD8⁺T cells and PD-1⁺CD8⁺T cells in all cases (r = 0.3724, p < 0.01). However, this inherent characteristic was only noticed in peripheral rather than central TME. These observations suggested that CD38^hiCD8⁺T cells not only constitute the majority of PD-1 expressing exhausted T cell subset, but complemented as a specific subset of exhausted CD8⁺ T cells in whole TME.

3.5 Peripheral TME located CD38^hiCD8⁺T cell predicted clinic response to anti-PD-1 therapy in NSCLC

Of the total cases studied, 24 patients were administered anti-PD-1 therapy. Guided by the preceding data, we subsequently delved into whether the quantification of CD38^hiCD8⁺T cells across various regions could emerge as a predictive biomarker for the response to anti-PD-1 therapy. Initially, our analysis revealed that responders exhibited a significantly elevated level of CD38^hiCD8⁺T cells in the peripheral TME compared to non-responders (52.15 ± 3.28% versus 35.55 ± 5.10%, P ≤ 0.01). However, regarding the central TME located CD38^hiCD8⁺T cell, no significant difference was observed between responders and non-responders (21.08 ± 5.48% versus 19.34 ± 4.81%, P > 0.05) (Fig. 5a).

Consistent with above findings, the ROC analysis depicted in Fig. 5b underscored the robust association between the proportions of peripheral TME located CD38^hiCD8⁺T cells and the response status. In detail, the optimal cut-off value for these CD38^hiCD8⁺T cell proportion was determined to be 49.95%. This cut-off point achieved remarkable accuracy (88.89%), specificity (100%), sensitivity (75%), along with a good diagnostic performance (AUC = 0.9375). Conversely, considering the central TME located CD38^hiCD8⁺T cell level to responding clinic response, the cut-off of 12.30% only yielded an accuracy of 72.73%, specificity of 85.71%, and sensitivity of 66.67%, while the corresponding AUC in the ROC curve was 0.7143. These findings collectively suggest that the fraction of CD38^hiCD8⁺T cells within the peripheral TME holds a stronger potential as a biomarker for predicting post-therapeutic outcomes.

3.6 Higher CD38^hiCD8⁺T cell located in peripheral TME represented deeper response to anti-PD-1 therapy

Next, the percentage change from baseline regarding the sum of target lesion diameters for each patient was used to generate waterfall plots (Fig. 6a). In eight out of nine responders, the proportion of CD38^hiCD8⁺T cells located in peripheral TME exceeded the defined cut-off value (**P < 0.01), Also, as depicted in Fig. 6b, a higher infiltration of CD38^hiCD8⁺T cells in the peripheral TME corresponded to a deeper clinical regression upon anti-PD-1 therapy (r=-0.6603, P < 0.05). Conversely, no such correlation was noted in the CD38^hiCD8⁺T cells located in central TME (r = 0.0456, P > 0.05). These findings further emphasize the significant potential of the CD38^hiCD8⁺T cell fraction in the peripheral TME as a biomarker for predicting post-therapeutic outcomes.

3.7 PD-1 blockade decreased the level of CD38^hiCD8⁺T cell in vitro

To visualize that the immunotherapy effect is indeed better in patients with higher CD38^hi expression, we stimulated single cells isolated from human lung cancer tissues with anti-PD-1 mAb in vitro for 72h to mimic clinic setting and carried out FCM analysis (Fig. 7a). As shown in Fig. 7b, anti-PD-1 significantly reduced the expression of CD38 on CD8⁺T cells, resulting in a significant decrease in the ratio of CD38^hiCD8⁺ T cells (p < 0.05), whereas no significant change of CD38^intCD8⁺T cells level (p > 0.05). Interestingly, more PD-1 mAb treatment-mediated depletion of CD38^hiCD8⁺T cells in vitro was observed in subject with higher CD38^hiCD8⁺T cells (Fig. 7c, r = 0.9041, P < 0.01). These data supported that the decrease of CD38^hiCD8⁺T cells mediated by PD-1 blockade favored effective anti-tumor immunotherapy, and CD38^hiCD8⁺T cells could be the target cells of ICB.

3.8 Analysis of CD38^hiCD8⁺T cells in ICIs-resistant lung cancer model

To further validate our previous conclusions, we constructed a ICIs-resistant lung cancer model and analyzed corresponded TME. Similarly, mouse CD38⁺CD8⁺T cells were also prominently displayed as the two clusters (Fig. 8a). Meanwhile, stronger compromised mitochondrial quality and activity was also observed in tumor‑infiltrating CD38^hiCD8⁺T cells, which was consisted with findings in human experiments (Fig. 8b).

3.9 Involvement of altered CD38^hiCD8⁺cells in reverse of immunotherapy resistance to PD-L1mAb

Epigallocatechin gallate (EGCG), the main constituent of green tea catechins, has proven its role in cancer management through modulating various mitochondrial metabolism pathways such as mitochondrial biogenesis, mitochondrial bioenergetics, mitochondria-mediated cell cycle and apoptosis. ^{[16, 17]}. Next, to explore whether the resistance to anti-PD-L1 mAb could be restored by intervening on CD38^hi/CD38^intCD8⁺T cells, we co-administered EGCG and anti-PD-L1 mAb to ICIs-resistant lung cancer model (Fig. 9a). As shown in Fig. 9b, treatment with EGCG or anti-PD-L1 mAb only partially inhibited the growth of tumors (P > 0.05), but more potent inhibition of tumor growth in mice was seen in the group treated with combinatorial therapy (P < 0.0001). Furthermore, mice treated with both EGCG and anti-PD-L1 mAb experienced the greatest survival benefit as compared to the others group (P < 0.0001, Fig. 9c).

To understand the impact of this therapy on the tumor microenvironment and determinate cell populations that contributed to delayed tumor growth and survival in treated mice, we analyzed TILs by flow cytometry. Alone anti-PD-L1 mAb or EGCG treatment had weekly effect on the ratio of infiltrating CD38^hiCD8⁺T cells, whereas the infiltrated degree of CD38^hiCD8⁺T cells was obviously decreased in the combinatorial treated groups (P < 0.0001, Fig. 9d). Notably, there was no difference between combinatorial therapy and other groups in terms of the ratio of infiltrating CD38^intCD8⁺T cells and CD38^negCD8⁺T cells (P > 0.05). These results indicated that altered CD38^hiCD8⁺T cells plays a direct role in reverse of immunotherapy resistance to PD-L1mAb.

3.10 Dual therapy improved mitochondrial by tumoral CD38^hiCD8⁺T cells

Having established the selective alteration in the quantity of tumoral CD38^hiCD8⁺T cells, we next determined whether the enhanced tumor control ability following combination therapy is also influenced by the function of CD38^hiCD8⁺T cells. Given mitochondrial translation selectively regulates the expression of a subset of proteins, including those involved in the cytotoxic T lymphocyte killer response, we examined mitochondria of CD38^hiCD8⁺T cells. Of note, as judged by expression of MTG, TMRE, Rhod-2 and Mito SOX (Fig. 10a-d), alone EGCG or anti-PD-L1 had no significant impact on mitochondrial activity in CD38^hiCD8⁺T cells (p > 0.05), only combination therapy improved mitochondrial metabolic activity of these CD8⁺T subset (*p < 0.001, **p < 0.0001, ***p < 0.001, **** p < 0.0001). Instead, combination therapy didn’t impact the mitochondrial activity by tumoral CD38^int/CD38^loCD8⁺T cells (p > 0.05). It was again indicated that the specially altered mitochondrial in CD38^hiCD8⁺ T cells might participate in reversing a therapeutic resistance for ICB.

3.11 Increase of IFN-γ secretion is accompanied by mitochondrial improvement in CD38^hiCD8⁺T cells

Naturally, it was found that the IFN-γ secretion in the fully activated condition induced by PMA and Ionomycin by CD38^hiCD8⁺T cells was significantly decreased compared with CD38^intCD8⁺T cells and CD38^negCD8⁺T cells (9.20 ± 1.93% vs 12.95 ± 1.97% vs 16.89 ± 2.51%), which means that CD38^hi marked a specific subset of CD8⁺ T cells with dysregulated cytotoxicity (Fig. 11a). However, it was revealed no significant correlation between the level of IFN-γ and expression of TMRE, Rhod-2 and Mito SOX by naïve CD38^hi/CD38^intCD8⁺T cells (P > 0.05, Fig. 11b).

By expanding data in vivo, dual therapy substantially increased the ability of IFN-γ secretion of CD38^hiCD8⁺T cells rather than monotherapy (Fig. 11c). Correspondingly, inherent linearity between improved mitochondrial metabolism and favorable IFN-γ secretion was only found in CD38^hiCD8⁺T cells from dual therapy group (Fig. 11d). These data provided evidence again that CD38^hiCD8⁺T cell is closely associated ICB mediated anti-tumor immunity, particularly on its altered mitochondrial activity, which serves a mechanistic role in rendering anti-PD-1 therapeutic resistance.

CD38, initially found on thymocytes and T lymphocytes, was considered as an activation molecule, involved in lymphocyte activation, proliferation, and adhesion^[18]. More importantly, as the indicator of functional feasibility, CD38 is a vital surface marker in CD8⁺ T cells, which can act as surface molecule by regulating the intracellular levels of calcium and downstream signaling pathways through its ADP-ribosyl cyclase activity^[14]. However, there are also some studies suggesting that CD38 can act as an immune checkpoint for T cells^{[2, 19]}. Besides, depletion of CD38⁺ immune regulatory cells resulted in an increase in T-helper cells, cytotoxic T cells, T-cell functional response and TCR clonality in multiple myeloma^[20]. Thus, CD38 may be a multifunctional molecule and the property of CD38 as a surface marker on CD8 + T cells has not been confirmed. Therefore, to identify the functional characteristics of CD38⁺CD8⁺ T cells in NSCLC is demanding.

In the present study, we demonstrated that CD38^hiCD8⁺ T cell subsets were selectively accumulated in peritumor rather than intratumor compartments. Meanwhile, another interesting result attracted our attention. The tumor-infiltrating CD38^hiCD8⁺ T cells have significantly expressed high level of PD-1 compared to paired CD38^intCD8 + T cells. What’s more, there is a strong positive relationship between peripheral TME located CD38^hiCD8⁺T cells and PD-1⁺CD8⁺T cells. This suggests that PD-1 expression on CD38^hiCD8⁺ T cells indicates an exhausted subset found in peritumor TME. Therefore, combined targeting of CD38 and PD-1 may be a novel immunotherapeutic approach.

Drugs targeting the PD-1/PD-L1 pathway have revolutionized the treatment of NSCLC with a subset of patients experiencing durable responses. However, still a majority of patients and cancer types do not, or only temporarily respond to these immune checkpoint blocking (ICB) drugs^{[5, 21]}. Thus, the search for novel biomarkers with improved response predictions is ongoing. Several studies have reported that CD8 levels combined with other signatures such as tumor mutation burden and PD-L1 expression, can predict responses to PD-1/PD-L1 blockade in patients with NSCLC, particularly when CD8 + T cells are densely accumulated in the invasive tumor margin^{[22, 23]}. Previous study demonstrated that CD38 is highly expressed on neoantigen-reactive CD8 T cells and PD-1 + CD38^hiCD8 + T cells serve as a predictive and therapeutic biomarker for PD-1 or PD-L1 blockade^[14]. However, our study provides an effective prediction method with a very high predictive power of CD38^hiCD8 + T cells for anti-PD-1 therapy with an AUC = 0.9. Therefore, we recognize that CD38^hi phenotype of CD8 + T cells can be a predictive and therapeutic biomarker of anti-PD-1 treatment as well as for selecting patients that would benefit from anti-PD-1 therapy.

Our observations reveal that the impairment of mitochondrial metabolic activity particularly existed in CD38^hiCD8 + T cells. These results were consistent with a recent study, which reported that the exhaustion states of CD8 + T cells are closely related to dysregulated mitochondrial integrity or activity^[24–26]. However, the impacts of these mitochondrial abnormalities on tumor infiltrating T cell function are largely unexplored. Mitochondria are involved in various aspects of CD8 + T cell function, including the regulation of translation of key effector function proteins and the control of the fate of CD8 + T cell differentiation^{[27, 28]}. Accumulation of depolarized mitochondria has been described as one of the causes of functional defect of tumor-infiltrating T cells, leading to impaired ability to kill tumor cells^[29]. We found a similar CD8 + T cell dysfunction caused by mitochondrial defects in CD38^hiCD8 + T cells of lung cancer. The altered mitochondrial dynamics of tumor-infiltrating T cells can be reversed by administering the NAD + precursor nicotinamide riboside, which acts synergistically with anti–PD-1 treatment in controlling tumor growth. CD38 has also been reported to cause age-related NAD + decline and cellular senescence, which further leads to mitochondrial dysfunction.

Herein, we aimed to characterize the unique subset of T cells that express CD38 in TME from the perspectives of mitochondrial metabolism and functional regulation. Our findings demonstrate the mechanistic role of CD38 in perturbing mitochondrial fitness, and this functional defect could be overcome by potential of genetic ablation or antibody-mediated targeting of CD38. These data offer additional avenues for biomarker and combination-immunotherapy discovery.

Ethics Approval and Consent to Participate

This study was approved by the Institutional Review Boards of The First Affiliated Hospital of Soochow University (2019-070). The processing of clinical tissue samples is in strict compliance with the ethical standards of the Declaration of Helsinki. Informed consent was obtained from all participants.

Availability of Data and Materials

The datasets used and analyzed during the current study available from the corresponding author on reasonable request.

Funding

The project was mainly supported by the National Natural Science Foundation of China (NSFC) grant 81874110 (to Q.Q), 81672280 (to C.C), Natural Science Foundation of Suzhou City grant SYS2021034 (to C.C).

Conflict of Interest

The authors declare that they have no competing interests.

Author Contributions:

Lei-lei Lv: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Writing – original draft, Writing – review & editing, Visualization. Jia-wei Zhai: Conceptualization, Methodology, Software, Investigation, Resources, Writing – review & editing. Jia-juan Wu: Conceptualization, Methodology, Investigation, Writing – review & editing. Gui-qin Fan: Methodology, Software, Investigation, Resources. Yao-xin Zhang: Methodology, Investigation, Writing – review & editing. Yu Shen: analysis, Resources. Qiu-xia Qu: Conceptualization, Methodology, Resources, Writing – original draft, Writing – review & editing, Supervision, Project administration, Funding acquisition. Cheng Chen: Conceptualization, Methodology, Resources, Writing – original draft, Writing – review & editing, Supervision, Project administration, Funding acquisition.

All authors reviewed and approved the final version to be submitted.

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No competing interests reported.

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High CD38 expression defines a mitochondrial function adapted CD8 + T cell subset with implications for lung cancer immunotherapy

Status:

Version 1

Abstract

Figures

Introduction

Material and methods

2.1 Patients

2.2 Sample collection

2.3 Mice and cell lines

2.4 Mouse tumor experiments

2.5 Analysis of tumor-infiltrating lymphocytes (TILs)

2.6 Mitochondrial analysis

2.7 In vitro blockade culture

2.8 Immunofluorescent Analysis

2.9 Assessment of Immunotherapy

2.10 Statistical analysis

Results

3.1 CD38⁺CD8⁺T cell divided into CD38^hi and CD38^int subsets in human lung cancer

3.2 Regional profile of CD38^hi/CD38^intCD8⁺ T cells in lung cancer microenvironments

3.3 Tumoral CD38^hiCD8 + T cells harbor more impaired mitochondria

3.4 CD38^hiCD8⁺T cells displayed a more exhausted phenotype

3.5 Peripheral TME located CD38^hiCD8⁺T cell predicted clinic response to anti-PD-1 therapy in NSCLC

3.6 Higher CD38^hiCD8⁺T cell located in peripheral TME represented deeper response to anti-PD-1 therapy

3.7 PD-1 blockade decreased the level of CD38^hiCD8⁺T cell in vitro

3.8 Analysis of CD38^hiCD8⁺T cells in ICIs-resistant lung cancer model

3.9 Involvement of altered CD38^hiCD8⁺cells in reverse of immunotherapy resistance to PD-L1mAb

3.10 Dual therapy improved mitochondrial by tumoral CD38^hiCD8⁺T cells

3.11 Increase of IFN-γ secretion is accompanied by mitochondrial improvement in CD38^hiCD8⁺T cells

Discussion

Declarations

References

Additional Declarations

Status:

Version 1

High CD38 expression defines a mitochondrial function adapted CD8 + T cell subset with implications for lung cancer immunotherapy

Status:

Version 1

Abstract

Figures

Introduction

Material and methods

2.1 Patients

2.2 Sample collection

2.3 Mice and cell lines

2.4 Mouse tumor experiments

2.5 Analysis of tumor-infiltrating lymphocytes (TILs)

2.6 Mitochondrial analysis

2.7 In vitro blockade culture

2.8 Immunofluorescent Analysis

2.9 Assessment of Immunotherapy

2.10 Statistical analysis

Results

3.1 CD38+CD8+T cell divided into CD38hi and CD38int subsets in human lung cancer

3.2 Regional profile of CD38hi/CD38intCD8+ T cells in lung cancer microenvironments

3.3 Tumoral CD38hiCD8 + T cells harbor more impaired mitochondria

3.4 CD38hiCD8+T cells displayed a more exhausted phenotype

3.5 Peripheral TME located CD38hiCD8+T cell predicted clinic response to anti-PD-1 therapy in NSCLC

3.6 Higher CD38hiCD8+T cell located in peripheral TME represented deeper response to anti-PD-1 therapy

3.7 PD-1 blockade decreased the level of CD38hiCD8+T cell in vitro

3.8 Analysis of CD38hiCD8+T cells in ICIs-resistant lung cancer model

3.9 Involvement of altered CD38hiCD8+cells in reverse of immunotherapy resistance to PD-L1mAb

3.10 Dual therapy improved mitochondrial by tumoral CD38hiCD8+T cells

3.11 Increase of IFN-γ secretion is accompanied by mitochondrial improvement in CD38hiCD8+T cells

Discussion

Declarations

References

Additional Declarations

Status:

Version 1

3.1 CD38⁺CD8⁺T cell divided into CD38^hi and CD38^int subsets in human lung cancer

3.2 Regional profile of CD38^hi/CD38^intCD8⁺ T cells in lung cancer microenvironments

3.3 Tumoral CD38^hiCD8 + T cells harbor more impaired mitochondria

3.4 CD38^hiCD8⁺T cells displayed a more exhausted phenotype

3.5 Peripheral TME located CD38^hiCD8⁺T cell predicted clinic response to anti-PD-1 therapy in NSCLC

3.6 Higher CD38^hiCD8⁺T cell located in peripheral TME represented deeper response to anti-PD-1 therapy

3.7 PD-1 blockade decreased the level of CD38^hiCD8⁺T cell in vitro

3.8 Analysis of CD38^hiCD8⁺T cells in ICIs-resistant lung cancer model

3.9 Involvement of altered CD38^hiCD8⁺cells in reverse of immunotherapy resistance to PD-L1mAb

3.10 Dual therapy improved mitochondrial by tumoral CD38^hiCD8⁺T cells

3.11 Increase of IFN-γ secretion is accompanied by mitochondrial improvement in CD38^hiCD8⁺T cells